Research in the area of the use of plant pathogens as biological control
agents for weeds is conducted using either the classical or the bioherbicidal
approach. In the classical approach, a pathogen is typically imported from a
foreign location to control an introduced weed target. The pathogen is applied
or released into a small weed population relative to the total infestation. This
is commonly a single application and if conditions are favorable, the pathogen
spreads throughout the target weed population. In the inundative or bioherbicide
strategy, an indigenous pathogen is cultured to produce large quantities of
inoculum that are applied at high rates to the entire target weed population.
There are hundreds of plant pathogens that have been tested for their potential
as bioherbicide candidates. Research on the development of plant pathogens for
biological control using the inundative or bioherbicide approach has moved from
determining host range and demonstrating pathogenicity to investigating systems
that enhance the efficacy of these agents.

Enhancing Bioherbicides

Formulation with water-retaining additives to reduce dew
dependence. The
requirement for long periods of dew or moisture is a major hurdle in the
development of foliar fungal pathogens into bioherbicides, mainly because
humidity and dew period duration can significantly limit disease initiation and
disease progress. The utilization of formulations that minimize the influence of
humidity is one approach to overcoming this restraint (5). The addition of an
invert oil emulsion to conidial suspensions of Colletotrichum truncatum,
a biocontrol agent for hemp sesbania (Sesbania exaltata), reportedly
resulted in 100% control in the absence of dew in the greenhouse. In the field,
the same formulation caused > 95% control, similar to the control achieved with
the chemical herbicide acifluorfen (8) (Fig. 1). Improved bioherbicidal
efficacy by the addition of oil emulsions, with no or little exposure to dew,
has been reported for several other bioherbicide pathosystems (1,4,55).
Different modes of action have been proposed for the activity seen with invert
emulsions, including phytotoxicity (4,48) (Fig. 2).

Broadening the spectrum of bioherbicides. While host specificity is
desirable in some situations, particularly in areas where crop and weed species
are closely related, there are situations where several species of problematic
weeds occur and broad-spectrum activity is required. This has been often cited
as a major limitation to the bioherbicide approach in cropping systems (28).

Fig. 3. (A) Spores of the biological control agent, Colletotrichum gloeosporioides, which was isolated from coffee senna (Senna occidentalis). (B) When combined with an oil-based formulation, the pathogen will also control sicklepod (S. obtusifolia). Photos courtesy of Doug Boyette.

Simultaneous control of northern jointvetch (Aeshynomene indica) and
winged waterprimrose (Jussiaea decurrens) was accomplished by applying a
combination of two host-specific pathogens, C. gloeosporioides f. sp.
aeschynomene and C. gloeosporioides f. sp. jussiae (11). A
mixture of three host-specific pathogens was used for simultaneous and
efficacious control of pigweed, sicklepod, and showy crotolaria (16). Excellent
control of seven grass species was also achieved when conidia of three pathogens
(Dreschelara gigantea, Exserohilum rostratum, and E.
longirostratum) were applied together (Fig. 4) (17).

Fig. 4. The multiple-pathogen strategy proposed by Chandramohan et al. (17) allows for the control of a broader spectrum of weeds than a single pathogen used alone and may address long-term concerns about the potential for resistance.

Another approach is to utilize pathogens that have a host-range that is not
as restricted. Myrothecium verrucaria was first evaluated for sicklepod
(53) and kudzu control (12), but has since been evaluated for multiple weed
targets (Fig. 5). However, the use of M. verrucaria is likely to be
limited due to its production of trichothecenes, although these toxins have not
been detected in treated plants (2). Additional research into the necessity of
trichothecenes for pathogenesis and their potential for mammalian toxicity when
the pathogen is used in a bioherbicide system is warranted.

Fig. 5. Impact of Myrotheccium verrucaria on a mixed stand of spurge and purslane species. The broad-spectrum pathogen had no affect on tomatoes later transplanted into the plots (Doug Boyette, personal communication). Photo courtesy of D. Boyette.

Enhancing bioherbicidal efficacy through delivery or application systems. Application of high volumes of propagules has been done to ensure complete
coverage of infection courts and to provide ample moisture needed for spore
germination for maximum infection (38). The requirement for high application
volumes for bioherbicides can deter their use because this entails transport of
a heavier load to the application site and longer application times. It has been
shown that high volumes do not necessarily ensure high levels of disease.
According to Lawrie et al. (40), the number of lesions produced or the infection
density is influenced not by the application volume alone but by the spray
droplet size, droplet retention and distribution, inoculum concentration, and
spray application volume. The type of spray equipment employed, as well as the
formulation and the inoculum concentration influences droplet size, droplet
retention, and distribution.

Chapple and Bateman (18) reported significant differences in the number of
deposited droplets that did not contain any spores in a spray equipment
comparison. The percentage of droplets without any spores was 67.3% for the
hydraulic flat fan, 95% for the air blast sprayer, and 6.5% for the spinning
disc. Yandoc (55) employed an air assisted sprayer versus a hand-held ultra low
volume sprayer (ULVA) to apply Bipolaris sacchari conidia (formulated in
an oil emulsion) to cogongrass (Imperata cylindrica) in the field and
found that application with the ULVA resulted in greater damage (> 50%) than with
the air-assisted sprayer (< 35%).

Another interesting approach to application technology involves the use of
the virus Tobacco mild green mosaic virus (TMGMV) for control of tropical soda
apple (Solanum viarum) (22). This weed is a problem in cattle pastures,
and control with a biological control agent requires applications over large
areas. The virus has a dramatic impact, causing complete plant death (Fig. 6).
Novel application methods have been effective and could easily be implemented by
ranchers. These include low pressure application (20 psi) of the virus combined
with plant abrasion using either a section of chain-link fence or carpet, and
high pressure application (400 psi) directly to plants (Fig. 7).

Fig. 7. Tropical soda apple is a problem weed on cattle ranches and its control requires coverage of large acreages. The use of the biological control agent Tobacco mild green mosaic virus (TMGMV) is feasible through the use of novel application approaches, including abrasion of plants while spraying the virus and high-pressure spraying but not with the mow-and-spray application.

Other ways of delivering bioherbicides, specifically solid-based
formulations, have been tested for pre-emergence bioherbicides that are meant to
attack weeds at or below the soil surface (9). Among the substrates used are
Pesta (wheat-gluten matrix) for C. truncatum, A. crassa, and
Fusarium lateritium (23) and F. oxysporum f.sp. orthoceras
(49), and cornmeal-sand for F. solani f. sp. cucurbitae, a
bioherbicide for Texas gourd (10). Formulation with solid substrates allows for
improved shelf life and also acts as a buffer when extreme conditions occur in
the field (6).

In another study, composted chicken manure supported the growth of
Trichoderma virens as well as its production of viridiol, a compound that
inhibits weed seed germination and emergence (34). In greenhouse tests, the
application of T. virens infested manure reduced emergence by 77% and dry
weight by 68% of naturally occurring miscellaneous weed species (34). Similarly,
the combination of an allelopathic cover crop, rye, with Trichoderma-inoculated
compost was used to control weeds in transplanted vegetables (29).

Enhancing biocontrol efficacy through selection and use of amino acid
excreting strains. Tiourebaev et al. (51) have attempted a novel approach to
enhancing virulence of weed biological control agents by selecting for strains
that are capable of excreting high levels of amino acids. Improved efficacy of
F. oxysporum f.sp. cannabis, a potential biocontrol agent for
Cannabis sativa was achieved with valine-excreting mutants. The mutants
failed to infect or cause damage to other plant species tested, indicating no
change in the host-range. Other weed-pathosystems that might employ this
approach are under investigation (52,58)

Combination of biocontrol agents with herbicides and other chemical agents
that predispose weeds to infection. One factor that can influence the level
of weed suppression through biological means is the ability of a target weed to
resist infection and colonization by the biological control agent. The
application of the biocontrol agent A. cassiae to sicklepod resulted in
increased activity of phenylalanine ammonia-lyase (PAL) (31), an enzyme
responsible for the synthesis of phenolic compounds that have been shown to
protect plants from pathogen attack. Hoagland (32) presented several approaches
for improving biocontrol efficacy by disrupting the target weedís defense
mechanisms, including the use of herbicides or other compounds that affect key
enzymes, blocking the synthesis of secondary plant metabolites, or breaking down
physical barriers to pathogen attack, all of which had encouraging results.

Enhanced bioherbicidal efficacy of E. monoceras on Echinochloa
crus-galli was observed when the pathogen was applied with δ-aminolevulinic
acid, a precursor of tetrapyroles, which are involved in the bleaching and
killing of plant tissue (30). Gressel et al. (26) demonstrated that the efficacy
of a weakly pathogenic agent, C. coccodes on velvetleaf (Abutilon
theophrasti), can be improved by applying chemical agents that repress host
plant defenses. A low dose of fungal inoculum applied with calcium chelators
resulted in increased infectivity by C. coccodes and reduced callose
formation. Hodgson et al. (33) reported 75 to 90% velvetleaf mortality from the
application of tank-mix combinations of C. coccodes and lower rates of
thidiazuron, a plant growth regulator. The mixes were almost twice as effective
as the thidiazuron applied alone (33).

The strategy of applying sub-lethal rates of glyphosate with conidia of A.
cassiae to improve the level of weed control has been tested. Sharon et al.
(50) demonstrated that the ability of glyphosate to inhibit the accumulation of
phytoalexin rendered sicklepod seedlings more susceptible to infection by A.
cassiae even at low inoculum concentration levels. Peng and Byer (44)
reported greater suppression of green foxtail (Setaria viridis) with a
mixture of low rates of sethoxydim and Pyricularia setariae as compared
to the level of control achieved with herbicide or pathogen alone. Several other
pathogens have been tested for their compatibility with crop protection
chemicals, including herbicides with which they might be tank-mixed (54,57) (Fig. 8).

Fig. 8. Fungi with potential for use as biological weed control agents in agro- ecosystems should be tested for compatibility with other pesticides that may be used in the system. Pictured here is a Petri plate assay of Dactylaria higginsii, a potential biological control agent for nutsedges, grown on media amended with pesticides that might be used in a conventional tomato production system (57).

Techniques for the Improvement of Weed Control Efficacy with Pathogens

Weed control through pathogen application and plant competition. The
impact of bioherbicides on the target weedís ability to compete has been well
demonstrated. An early example of this phenomenon is Puccinia chondrillina,
a rust specific to skeletonweed (Chondrilla juncea), which inhibited that
plantís ability to compete with subterranean clover (Trifolium subteraneum)
(27). Velvetleaf growth was stunted and seed production was reduced by the
presence of C. coccodes, rendering it less competitive with soybean (25).
Dactylaria higginsii applied to mixed plantings of tomato and purple
nutsedge reduced interference from this weed and increased the yield of tomato
(36).

Application of Bipolaris sacchari to mixed plantings of cogongrass and
bahiagrass (Paspalum notatum) resulted in reduced cogongrass growth and
allowed bahiagrass to flourish (56). This strategy involves the specific
suppression of a weedy grass species while allowing for a beneficial grass
species to establish and take over the niche vacated by the weed and prevent its
re-establishment (Fig. 9).

Fig. 9. (A) Selective control of cogongrass (Imperata cylindrica) by Bipolaris sacchari, formulated in an invert emulsion, in a mixed planting of the weed in bahiagrass (Paspalum notatum). (B) Control of cogongrass, as seen in the pot on the left side, allows bahiagrass to flourish when compared to the uninoculated control on the right.

Although it would seem logical that weed interference could be reduced by
competition from increased crop plant density coupled with bioherbicide
applications, Pitelli et al. (45) did not find evidence to support this
hypothesis. In their study, soybean planting density did not impact the
mortality or the dry weight of sicklepod sprayed with A. cassiae and/or
Pseudocercospora nigricans. While similar studies of
crop-weed-bioherbicide combinations are not common, the need for this type of
competition study is necessary to characterize possible avenues for enhanced
control using integrated weed management systems that include bioherbicides.

Biocontrol and host plant resistance. A novel method of utilizing
non-target plant competition was tested for management of striga (Striga
hermonthica) (43). F. oxysporum was applied in each planting hole
before sowing seeds of the resistant sorghum cultivar Samsorg 40 and the
tolerant landrace Yaríruruka. Plots treated with F.
oxysporum and planted to each of the cultivars had significantly higher crop
yield and lower striga infestation compared to plots that were planted with the
same cultivars but not treated with F. oxysporum.

Deleterious rhizobacteria and cover crops for improved weed control. The
potential of deleterious rhizobacteria (DRB) to serve as control agents of weeds
relies on their ability to reduce weed emergence and to delay growth and
development, giving the crop a competitive advantage (6). Reduced downy brome (Bromus
tectorum) population and 18 to 35% yield increase in winter wheat through the
application of the DRB Pseudomonas fluorescens has been reported (35,37).
However, there are also reports of inconsistent performance by DRB, due to poor
survival or low activity once applied in the field. An integrated approach
utilizing DRB and cover crops for improved weed control was also tested (41).
The cover crops support the proliferation and activity of the DRB before weed
seed germination occurs, which enhances the level of weed suppression by the
cover crops. The DRB-cover crop combination caused greater weed biomass
reduction compared to cover crops alone. The characterization of cropping
systems that support microbial populations that are weed-suppressive is an
emerging area of research that shows great promise (24).

Combining pathogens and insects. Management of leafy spurge (Euphorbia
esula-virgata) in natural areas has been problematic because chemical
herbicides have been ineffective or inappropriate and leafy spurge can easily
overcome damage from mechanical weed control measures and insect biocontrol
agents (42). While the release of root-feeding flea beetles (Aphthona
spp.) resulted in successful insect establishment, the insects did not
significantly reduce the weed population (13). The frequent presence of
soilborne pathogens at release sites where leafy spurge is in decline has
spurred interest in combining soilborne pathogens isolated from leafy spurge
roots and flea beetles for a more effective control (Fig. 10) (14). This
approach may be possible for the control of white top (Cardaria draba),
as plants with insect damage also harbor pathogens that contribute to the
decline of plant stands (Anthony Caesar, personal communication) (Fig. 11).
Interestingly, these interactions are even more complex than they initially
appear. Pathogens associated with insect-infested weeds have been found to be
more highly virulent than the same species of fungi isolated from diseased weeds
that are not infested with the insects (15,39). A three-way interaction has also
been found among deleterious rhizobacteria, fungal plant pathogens and insects
(39).

Fig. 10. (A) Field location with a significant leafy spurge (Euphorbia esula) population prior to the release of the flea beetle Aphthona flava and (B) four years after the release. (Photo credit Norman Rees, ARS, courtesy of Tony Caesar). (C) At several sites where the weed stand was reduced, soilborne pathogens were found to be associated with the affected plants. Photo courtesy of Tony Caesar.

The strategy for controlling weeds using the combination of insects and plant
pathogens is supported by reports of population regulation resulting from damage
from natural enemies (19,21). The rust Puccinia psidi is currently under
investigation as a biological control agent for Australian melaleuca (Melaleuca
quinquenervia) (46). Researchers in this area (Min Rayamajhi, personal
communication) reported significant reduction in the absolute density and
diameter based density of the Australian melaleuca in the presence of and during
an increase in the population of natural enemies (i.e., weevils, rust, lobate
lac scales, psyllids, and sooty molds) (Fig. 12). The impact of these pests is
dramatic and allows for the regeneration of the understory (Fig. 13). The
elucidation of the interactions between these natural enemies is the current
area of research (47).

The effort to develop plant pathogens as commercially available bioherbicides
has not yielded the large number of products that the research would indicate.
However, research in this area has nonetheless contributed significantly to the
science and technology of biological control. The development of this
weed-control technology, albeit used to a very limited extent, has resulted in
several commercial products, including four pathogens registered within the past
five years. Furthermore, as Hallett (28) points out, there are opportunities for
the development of bioherbicides for some specialized niches, such as parasitic,
urban, and allergenic weeds. For example, Smolder (A. destruens), has
been registered recently for the control of several Cuscuta (dodder)
species.

It is true that with the current state of the art, it is difficult for
companies to sustain business with a single or few registered bioherbicide
products. However, it is not a lack of proven efficacy that has limited the
availability of these materials; it is the market-driven return on investment
that is the constraint. For example, both DeVine and Collego are extremely
effective materials, but according to Dave Goulet of Encore Technologies
(personal communication) the market was too small and sporadic to maintain the
economic viability of the products for his company. Fortunately, a new
registrant, ARI Incorporated, has taken on the marketing of C.
gloeosporioides f. sp. aeschynomene (the Collego pathogen), as the
commercial product LockDown for use in rice in Arkansas, Louisiana, and
Mississippi (Dave TeBeest, personal communication). In the developed world, the
goal of most single-pathogen/single-weed bioherbicide research projects is a
saleable product. Therefore, the long-term success of bioherbicides may depend
on wholesale changes in consumerism and production systems that demand
non-chemical approaches. Differences in regulatory requirements among countries
may allow for some systems to gain greater acceptability in places where local
production of inoculum for immediate use is realistic. In short, there are still
stakeholders that would benefit from the bioherbicide technology (20).

Research on weed-pathogen systems has greatly contributed to knowledge in
plant disease epidemiology, plant-microbe interactions, and biodiversity. This
knowledge now provides the basis to understand and study multi-trophic
interactions in weed-pathogen systems. Many of the problematic weeds are often
found in monocultured crops or they exist in crops where low-cost weed control
is critical. Although most biological control agents are too host-specific to
individually address mixed weed populations in agronomic field crops, they can
be targeted to manage those weeds that have the most impact on crop yield in
high-value crops where control options are limited. Some cropping systems will
inherently favor a single dominant weed species, leading essentially to a
monoculture in which a host-specific biological control agent is ideally suited.
Organic and conventional vegetable and herb production systems are particularly
well suited to this approach and are in dire need of weed control options.
Continuing to place emphasis on the development of integrated weed control
systems that employ plant pathogens as components can greatly increase the
number of successful biological weed control programs.

Disclaimer

Mention of a trademark, warranty, proprietary product or vendor does not constitute a guarantee by the United States Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable.